Human papillomavirus (HPV) is a sexually transmitted virus and known precursor to cervical cancer, the second most lethal cancer in females across the world. Two virus-like particle (VLP) vaccines exist that provide immunity against the main serotypes of the disease and are produced in Saccharomyces cerevisiae (S. cerevisiae) and baculovirus infected insect cells. Pichia pastoris (P. pastoris) was chosen as an alternative expression system for HPV VLP production based on its history as prolific heterologous protein producer that circumvent many of the problems associated with aforementioned expression systems. The strongly inducible AOX promoter allows three-phase fermentations (1.3 L bioreactors) in which high cell densities (>100gCDW.L-1) are obtained prior to induction with methanol. During the induction phase the dissolved oxygen concentration may be used to control addition of methanol. It is also possible to use predetermined methanol feed rates and to adjust the amount of additional oxygen sparged to maintain a constant dissolved oxygen level. The effects of these control strategies, different gene constructs and multiple gene integrations were quantified through monomer-, VLP- and mRNA production levels. Increased biomass concentrations in the 20% dissolved oxygen control strategy led to the highest volumetric VLP concentration (68.53 mg.L-1). VLPs were located intracellularly in both the cytoplasm and membranes of the yeast cells. Despite lower codon adaptation of the h-L1 gene expressed in the X33[h-L1] strain it still had higher volumetric VLP concentrations under 40% dissolved oxygen control than the X33[Syn-L1] and X33[SA-L1] strain containing the SA-L1 and Syn-L1 genes. This was ascribed to the possible presence of rare codons in the Syn-hL1 and SA-L1 genes and a lower A+T content in the h-L1 gene. Multiple gene integrations of the h-L1 gene had a negative effect on VLP production and this conclusion was supported by lower mRNA concentrations indicating lower transcriptional efficiency. Increased methanol induction efficiency in the DO control strategies was indicated by higher specific L1 monomer levels. Decreased VLP to monomer ratios in the DO control strategies indicated that a bottleneck existed in the assembly process due to increased L1 monomer concentrations. Due to the hydrophobic region on the L1 protein, these proteins associated with the membranes within the yeast cells especially when efficient assembly to VLPs did not occur. HPV16 L1 VLP concentrations obtained in P. pastoris in this study are comparable to the study by Li et al., (2003), but much lower than expression levels obtained in baculovirus infected insect cells. Based on the expression levels of HBsAg VLPs obtained in P. pastoris, this system, with the necessary recommended optimisation, has the capacity for increased HPV VLP production ability.

Introduction: Human papillomavirus (HPV) is the most commonly occurring sexually transmitted infection and is a necessary cause of cervical cancer. The progression from HPV infection to cervical cancer is incompletely understood. Innate immune response to HPV infection has recently been identified as a potential cofactor in this progression. This study examined potential association(s) between killer cell immunoglobulin-like receptors (KIR) and HPV infection. HPV concordance was estimated among heterosexual couples demonstrating the complexity of HPV infection.Methods: HPV concordance was cross-sectionally estimated in 29 heterosexual couples. A polymerase chain reaction based assay for KIR genotyping was developed and validated. 283 women from the Young Women's Health Study and 259 men from the HPV Infection in Men: A Prospective Cohort Study had HPV infection data and samples available for KIR genotyping. Associations between KIR genotype and haplotype with HPV prevalence, incidence and clearance were assessed.Results: Among 29 couples, prevalence for any HPV type was comparable between women 86.2% and men, 75.9%. Partial concordance was observed in 66% of the couples. Forty-one percent (41%) of couples had perfect concordance. A high degree of concordance was observed, however HPV type distributions differed in men and women. In women from the YWHS, KIR2DS5 was significantly associated with oncogenic HPV prevalence (Odds ratio [OR]: 0.56, 95% Confidence Interval [CI]: 0.31-0.99). Any HPV incidence was significantly associated with KIR2DL2 (Hazards Ratio [HR]: 2.11, 95% CI: 1.0-4.44), KIR2DS2 (HR: 2.44, 95% CI: 1.13-5.24), KIR2DS3 (HR: 2.36, 95% CI: 1.16-4.81), and KIR haplotype B (HR: 2.48, 95% CI: 1.02-6.02). Women lacking KIR2DS5 had an increased risk of any HPV acquisition in the presence of KIR2DL2 (HR: 2.95, 95% CI: 1.28-6.86), KIR2DS2 (HR: 3.33, 1.39-7.99), or KIR2DS3 (2.77, 95% CI: 1.24-6.19). In Men, KIR2DS3 was significantly associated with increased probability of any HPV clearance (HR: 1.91, 95% CI: 1.04-3.49).Conclusions: This research contributes to our understanding of HPV infection dynamics through the assessment HPV type concordance in sexual partners. Additionally, through the development of an assay for KIR genotyping, we were able to identify associations with KIR gene positivity and HPV prevalence, incidence, and clearance in men and women.

Purpose: This study investigates knowledge about HPV and examines if pediatric dental providers should include HPV education for guardians of patients 10-18 years. Methods: Legal guardians of 10-18 year-old patients of the Virginia Commonwealth University Pediatric Dental Clinic were enrolled in this prospective cohort study. Participants completed a baseline survey, were provided HPV education, completed an initial follow-up survey, and then completed a 6-month follow-up survey. Results: A total of 54 participants completed the baseline and initial follow-up surveys and 17 completed the 6-month follow-up survey. The average number of correct responses was 3.4 of 6 knowledge questions, which significantly improved to 5.4 at follow-up (P<.0001). The greatest increase in the percent responding correctly was regarding HPV and oropharyngeal cancer from 22% baseline to 91% at initial follow-up (P<.0001). Regarding Stage of Change, 14 (23%) of those not initially in the Action group had improved at least 1 stage. At the 6-month follow-up, 3 (43%) guardians reported completing the HPV vaccine series. Conclusions: These results demonstrate limited knowledge about HPV and highlight the pediatric dental provider’s ability to educate. Since the greatest knowledge gap pertained to HPV and oropharyngeal cancer, it is important for pediatric dental providers to increase their role in HPV education. As oral cancers are the purview of dentists, practitioners should be involved with their patients’ consideration of the HPV vaccine.

Human Papillomaviruses (HPVs) are small DNA viruses which specifically infect keratinocytes at different body sites. An association between cutaneous Squamous Cell Carcinoma (SCC) formation, UV irradiation and infection with a high-risk subset of cutaneous HPVs has been postulated although the underlying molecular mechanisms by which HPV may play a role in SCC development are not yet fully elucidated. Expression of the viral E6 oncoprotein has been shown to interfere with DNA damage responses and inhibit UV induced apoptosis, suggesting HPV can contribute to early stages in tumourigenesis. Here, expression of E6 from HPV types 5, 8, 10, 18 and 77 was shown to reduce UV- or Fas-induced apoptosis, and the changes in a range of intracellular apoptotic regulators were investigated. Additionally, the subject of cutaneous SCCs, in contrast to HPV-associated anogenital cancers, not harboring HPV DNA in every tumor cell was explored. Results herein show that expression of E6 from skin cancer-associated HPV types 5 and 8 induced the secretion of factors that were able to inhibit UV-induced apoptosis in non-HPV expressing cell lines and primary human keratinocytes. The anti-apoptotic effect of HPV E6 expression was found to be mediated in part by upregulation of Osteoprotegerin (OPG) and Interleukin 6 (IL6). Purified OPG and IL6, when added to cells together, but not individually, reduced apoptosis following UV irradiation. Evidence is shown that OPG and IL6 inhibit the extrinsic and intrinsic apoptotic pathways respectively. Furthermore immunohistochemistry of HPV-typed SCC sections shows that IL6 protein is up-regulated in HPV positive tumors compared to HPV-negative cancers. To further test the effects of HPV5E6 expression, in combination with UV irradiation, on primary human keratinocytes microarray studies were performed. These findings support the hypothesis that a small number of HPV infected cells influence UV induced apoptosis in the skin and contribute to tumourigenesis.

This dissertation examines vaccination rates for Human Papillomavirus (HPV) among college women 18-26 years of age who participated in the American College Health Association’s National College Assessment (ACHA-NCHA). Utilizing secondary data, this research sought to report HPV vaccination rates among a racially diverse population and to identify potential barriers to vaccination. The ACHA-NCHA survey provided a large sample size (N=68,193) in which to perform a binary logistic regression analysis. Demographic characteristics were analyzed as potential barriers to HPV vaccination. Additionally, lack of certain health behaviors were explored as potential barriers to HPV vaccination. In this study, White/non-Hispanic women had a higher HPV vaccination rate when compared to minority women. The binary regression analysis demonstrated that minority women were less likely to receive the HPV vaccine. Furthermore, it was determined that as the age of the respondents increased, the likelihood of receiving the vaccine decreased. Health behaviors that were predictive of receiving the HPV vaccine included receiving the Hepatitis B and Influenza vaccine, number of sexual partners and receiving sexually transmitted disease information. Women who received a gynecological exam were almost twice as likely to receive the vaccine, as were women who had parental health insurance coverage. One aim of The Affordable Care Act (2010) is to decrease disparities in health care. Drawing attention to potential barriers to HPV vaccination allows policy makers to make informed decisions regarding future activities to reduce disparities. Health promotion activities should be targeted to specific populations in an effort to increase HPV vaccination rates.

Le cancer du col de l’utérus est la première cause de cancer chez la femme en Afrique de l’Ouest, une région du monde où le virus de l’immunodéficience humaine (VIH) sévit de manière endémique. Ce travail s’inscrit dans le cadre de l’étude du lien entre ces deux pathologies ainsi que des spécificités du dépistage du cancer du col dans le contexte de l’infection à VIH.Notre travail de recherche a été conduit en plusieurs étapes. Une enquête hospitalière a tout d’abord comparée la fréquence du VIH chez des femmes atteintes de cancer du col et chez des femmes atteintes d’autres cancers. Nous avons ensuite mis en place un programme de dépistage des cancers du col par inspection visuelle au sein de trois cliniques VIH à Abidjan offrant cette intervention pendant une période de plusieurs mois. Un échantillon de ces femmes dépistées a enfin été prélevé pour la recherche de papillomavirus humains (PVH). Sur les 152 cas de cancer du col inclus dans la première enquête, 25% étaient VIH-positifs contre 4,7% chez les 257 patientes du groupe de comparaison, donnant un Rapport de Côte (RC) ajusté de 7,6 (3,6 – 16,2) pour l’association entre ces deux morbidités sévères. Un total de 4 046 femmes a été dépisté par inspection visuelle. La fréquence d’un test positif était de 9,0% (8,0 – 10,0) chez les 2 998 femmes VIH-positives et 3,9% (2,7 – 5,1) chez les 1 048 femmes VIH-négatives. La prévalence de l’infection à PVH oncogène était de 33,0% chez les 191 femmes VIH-négatives et de 52,8% chez les 254 femmes VIH-positives ayant pu être testé par PCR. Un taux de CD4<200 cellules/mm3 était associé à la présence d’un PVH oncogène (RC= 2,8 [1,1 – 8,3] Ref. CD4 ≥500). L’infection à VIH est fortement associée au risque de cancer du col ainsi qu’à la présence de ses précurseurs que sont les PVH. La mise en place de programmes de dépistage associé à une bonne reconstitution immunitaire semble être des mesures essentielles pour réduire le fardeau de ce cancer chez les femmes VIH-positives en Afrique de l’Ouest à l’ère de l’accès élargi aux antirétroviraux. / Cervical cancer is the leading cause of cancer among women in West Africa, where infection with the Human Immunodeficiency Virus (HIV) is endemic. This work study the link between these two pathologies as well as the specificities linked to cervical cancer screening in the context of HIV infection. Our research project was conducted in several stages. A first hospital-based study compared the prevalence of HIV in women with cervical cancer and in women with other cancers. We then implement a cervical cancer screening program with visual inspection methods in three HIV clinics in Abidjan during several months. A sample of women screened was finally selected and collected for human papillomavirus (HPV) identification. Of the 152 cases of cervical cancer included during the first study, 25% were HIV-positive compared to 4.7% among the 257 patients of the comparison group, giving an adjusted odd ratio (OR) of 7.6 (3.6 - 16.2). A total of 4,046 women were screened by visual inspection. The frequency of a positive test was 9.0% (8.0 - 10.0) in the 2,998 HIV-positive women and 3.9% (2.7 - 5.1) in the 1,048 HIV-negative women. The prevalence of oncogenic HPV was 33.0% in the 191 HIV-negative women and 52.8% in the 254 HIV-positive women that underwent PCR testing. A CD4 count <200 cells/mm3 was associated with the presence of oncogenic HPV (OR = 2.8 [1.1 - 8.3] Ref. CD4≥500). HIV infection is strongly associated with cervical cancer and the presence of its precursors, oncogenic HPV. The implementation of adapted screening programs combined with good immune reconstitution seems to be key measures to reduce the burden of cervical cancer in HIV-positive women in West Africa in the era of expanded access to antiretroviral drugs.

O Papilomavírus Humano (HPV), da família Papilomaviridae, são vírus epiteliotrópicos que provocam lesões de pele ou mucosa. O carcinoma do colo uterino é uma das principais causas de morte de mulheres em todo o mundo, sendo a infecção pelo HPV o principal fator de risco para o desenvolvimento do carcinoma cervical. Durante o processo maligno, a migração descontrolada de células neoplásicas, é uma característica fundamental da invasão tumoral, sendo que as metaloproteinases de matriz (MMPs) participam largamente deste processo pois são responsáveis pela clivagem de proteínas da matriz extracelular. O projeto propôs investigar a expressão de MMP-9 total em amostras cérvico-vaginais com inflamação, lesão e tumor. Foram coletadas duas amostras cérvico-vaginais como esfregaços para diagnóstico citológico e imunocitoquímica. A coleta foi realizada em 630 pacientes da Penitenciária Feminina de Sant´ana, no período de agosto de 2009 a agosto de 2010. As lâminas foram submetidas à imunocitoquímica para detecção da expressão de MMP-9. Os resultados indicaram um aumento da expressão de MMP-9 total diretamente proporcional com o aumento do grau das lesões nos esfregaços, corroborando com dados de histologia da literatura. Portanto, tal método de correlação de MMP-9 total com esfregaços cérvico-vaginais poderá auxíliar o prognóstico em esfregaços. / The Human Papillomavirus (HPV) are epitheliotropic viruses from family Papillomaviridae, which cause skin or mucous lesions. Carcinoma of the cervix is a major cause of death in women worldwide, and HPV infection a major risk factor for development of cervical carcinoma. During the malignant process, cell migration is a fundamental characteristic of tumor invasion, and the matrix metalloproteinases (MMPs) are responsible for the cleavage of extracellular matrix proteins. This project aims to investigate the expression of MMP-9 in different degrees of injury in the smear. We collected two cervical smears for cytological diagnosis and immunocytochemistry. Data collection was conducted in 630 women of the Sant´ana Women\'s Penitentiary, during the period August 2009 to August 2010. The slides were submitted to immunocytochemistry to detect the expression of MMP-9 and noticed an increase in protein expression in parallel with the increased lesions grade, which corroborates the histology of the literature and could be a method prognostic aid in vaginal smears.

O câncer cervical é a consequência mais séria da infecção por Papilomavírus humano - HPV, constituindo uma das principais causas de morte entre mulheres no mundo, remetendo a um importante desafio para a saúde pública mundial. Desenvolvemos a produção de VLPs contendo as proteínas L1 e L2 de HPV16, utilizando células epiteliais humanas 293-F em suspensão e em meio isento de soro fetal bovino. As células possuem metabolismo intenso, adequado para a expressão de proteínas heterólogas. Apresentam vantagens como a facilidade de cultivo, transfecção e ocorrência de processos como a glicosilação de proteínas, fosforilação, formação de pontes dissulfeto e outras modificações pós-traducionais essenciais para a função das proteínas, facilitando a produção similar às condições in vivo. O sistema de produção de VLPs L1L2 de HPV16 foi estabelecido de maneira eficiente, em células epiteliais humanas em suspensão e com resultados promissores, podendo contribuir para o desenvolvimento de uma vacina profilática de amplo espectro de proteção, com custo reduzido de produção. / Cervical cancer is the most serious consequence of infection by human papillomavirus - HPV, constituting one of the leading causes of death among women worldwide that points to a major challenge to global public health. We have developed the production of VLPs containing L1 and L2 proteins of HPV16, using human epithelial cells 293-F suspended in medium without fetal bovine serum. The cells have intense metabolism, suitable for expressing heterologous proteins. Present advantages such as ease of culture, transfection and occurrence of processes such as protein glycosylation, phosphorylation, formation of disulfide bonds and other essential post-translational modifications to protein function, facilitating the production similar to the conditions in vivo. The VLPs L1L2 of HPV16 production system was established efficiently in human epithelial cells in suspension and with promising results, which may contribute to the development of a prophylactic vaccine broad protection spectrum with reduced production cost.

Thesis advisor: Junona Moroianu / The L2 minor capsid protein of human papillomavirus is one of two structural proteins that comprise the icosahedral shell. Two potential, leucine-rich nuclear export signals (NESs) had been identified in the HPV16 L2 sequence, one in the n-terminus (51MGVFFGGLGI60) and one in the c-terminus (462LPYFFDSVSL471). DNA primers for mutant L2 proteins were designed to specifically target these two potential NES regions. Two primers had mutations in the n-terminal located NES (nNES), while the other two primers had mutations in the c-terminal NES (cNES). L2 nuclear retention mutants, RR297AA (“MS4”) and RTR313AAA (“MS5”), served as the templates for these NES mutations. Using mutagenesis, the desired secondary mutations were introduced into the mutant L2 genes in order to create four, distinct mutants: RR297AA + P463_ (“MS4 T1”), RR297AA + V469_ (“MS4 T2), RTR313AAA + P463_ (“MS5 T1”), and RTR313AAA + V469_ (“MS5 T2”). In contrast to the pancellular localization of the MS4 and MS5 L2 mutants, the “MS4 T1,” “MS4 T2,” “MS5 T1”, and “MS5 T2” mutants were all localized nuclearly. These results suggest that deletion of the cNES inhibits nuclear export of the HPV16 L2 minor capsid protein. / Thesis (BS) — Boston College, 2011. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology Honors Program. / Discipline: Biology.

Thesis advisor: James Lubben / In 2011, an estimated 12,710 women suffered from cervical cancer and 4,290 died from it in the U.S. HPV vaccination (HPV-V) and PAP screening (PAP-S) could reduce this burden. Using 2010 National Health Interview Survey data, current disparities in the use of PAP-S and HPV-V in U.S. women aged 18-30 years were investigated. An adapted Behavioral Model of Health Care Utilization guided the study. Main outcomes were PAP-S in prior year and ever-HPV-V, both initiation and completion. Adjusted predictor estimates were obtained through multivariate logistic regressions with appropriate statistical procedures and weights for complex survey design. A sub-analysis focused on unvaccinated women. The sample had 3,129 women aged 18-30 years, representing about 27 million women of similar age in the U.S. PAP-S, HPV-V initiation and completion rates were 53.5%, 17.9%, and 10.3%, respectively. Hispanics were 33% less likely than Non-Hispanic-Whites to initiate HPV-V. Non-Hispanic-Blacks were 55% more likely and 57% less likely than Non-Hispanic-Whites to receive PAP-S and complete HPV-V, respectively. Non-Hispanic Asians were 36% less likely than Non-Hispanic-Whites to receive PAP-S, but this result was borderline significant. Younger age and being unmarried were predictors of lower PAP-S but higher HPV-V. Ever gave birth was a predictor of higher PAP-S but lower HPV-V. Preventative behaviors (PAP-S and flu vaccination) were predictors of higher HPV-V. STI-history was a predictor of higher HPV-V and PAP-S. Not having health insurance for over one year or recent health provider visit were predictors of lower PAP-S and HPV-V. Living in the South was a predictor of lower HPV-V. Household income was not a predictor of any outcomes. Most common reported reason for no HPV-V was "no need." Study findings indicate interventions to mitigate disparities in cervical cancer prevention are needed. Tailored education interventions for both women and health care providers along with opportunities associated with the 2010 Affordable Care Act, such as broader access to health care, emphasis on health information technology, and initiatives with PAP screening and adult vaccination as potential quality indicators for performance/payment, can reduce these disparities. Future research should focus on the feasibility of alternative venues for receiving HPV-V and PAP-S. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Social Work. / Discipline: Social Work.